Internalization of CD40 regulates its signal transduction in vascular endothelial cells

The CD40 ligand (CD40L)-CD40 dyad can ignite proinflammatory and procoagulatory activities of the vascular endothelium in the pathogenesis and progression of atherosclerosis. Besides being expressed on the activated CD4(+) T cell surface (mCD40L), the majority of circulating CD40L reservoir (sCD40L) in plasma is released from stimulated platelets. It remains debatable which form of CD40L triggers endothelial inflammation. Here, we demonstrate that the agonistic antibody of CD40 (G28.5), which mimics the action of sCD40L, induces rapid endocytosis of CD40 independent of TRAF2/3/6 binding while CD40L expressed on the surface of HEK293A cells captures CD40 at the cell conjunction. Forced internalization of CD40 by constitutively active mutant of Rab5 preemptively activates NF-kappaB pathway, suggesting that CD40 was able to form an intracellular signal complex in the early endosomes. Internalized CD40 exhibits different patterns of TRAF2/3/6 recruitment and Akt phosphorylation from the membrane anchored CD40 complex. Finally, mCD40L but not sCD40L induces the upregulation of proinflammatory cytokines and cell adhesion factors in the primary human vascular endothelial cells in vitro, although both forms of CD40L activate NF-kappaB pathway. These results therefore may help understand the molecular mechanism of CD40L signaling that contributes to the pathophysiology of atherosclerosis.     

Gastric cancer cell lines AGS before and after CD40 signal activating

The aim of this study was to investigate the molecular mechanisms underlying the antitumour effects of CD40L through analysing the change of genes expression profile in AGS using Affymetrix Gene Chip. Human gastric carcinoma AGS cells were first incubated with 2 μg/ml sCD40L or equal volume of medium (control) in F12 medium. RNA was isolated from AGS and were reverse transcribed, labeled with digoxigenin-11-dUTP, and then hybridized with Clontech Atlas mouse cDNA expression arrays for comparison. Performing clustering analysis, we found that 7 detected genes were down-regulated and 38 were upregulated as the sCD40L acted on AGS. To further verify the results of gene chip screening, Gene Database was searched, finding that the most significantly up-regulated genes were Gadd45a, c-Jun and Bcl-2, and the most significantly down-regulated genes were Cyclin D1, CDC6, TNFR10B, c-IAP2 and ORC5L. Based upon these findings, the signalling pathways that possibly mediate CD40-induced apoptosis are proposed.     

Porphyran induces apoptosis related signal pathway in AGS gastric cancer cell lines

Porphyrans, the sulfated polysaccharides, are the main components of Porphyra. The potential apoptotic activities of porphyran were evaluated using AGS human gastric cancer cells. Porphyran did not affect the growth of normal cells, but did induce cancer cell death in a dose-dependent manner. The addition of 0.1% porphyran also reduced DNA synthesis after 24 h of exposure, suggesting that porphyran inhibits cancer cell growth by both decreasing cell proliferation and inducing apoptosis. AGS cells treated with porphyran displayed a marked increase in poly(ADP-ribose) polymerase (PARP) cleavage, as well as caspase-3 activation. The ability of porphyran to promote apoptosis may contribute to its usefulness as an agent capable of significantly inhibiting cell growth in AGS human gastric cancer cells. Insulin-like growth factor-I receptor (IGF-IR) phosphorylation was decreased in porphyran-treated AGS cells compared to control cells, which correlated with Akt activation. Thus, porphyran appears to negatively regulate IGF-IR phosphorylation by causing a decrease in the expression levels in AGS gastric cancer cells, and then inducing caspase-3 activation.     

TRAF6 is required for TRAF2-dependent CD40 signal transduction in nonhemopoietic cells

The emerging role of CD40, a tumor necrosis factor (TNF) receptor family member, in immune regulation, disease pathogenesis, and cancer therapy necessitates the analysis of CD40 signal transduction in a wide range of tissue types. In this study we present evidence that the CD40-interacting proteins TRAF2 and TRAF6 play an important physiological role in CD40 signaling in nonhemopoietic cells. Using mutational analysis of the CD40 cytoplasmic tail, we demonstrate that the specific binding of TRAF2 to CD40 is required for efficient signaling on the NF-kappaB, Jun N-terminal protein kinase (JNK), and p38 axis. In fibroblasts lacking TRAF2 or in carcinoma cells in which TRAF2 has been depleted by RNA interference, the CD40-mediated activation of NF-kappaB and JNK is significantly reduced, and the activation of p38 and Akt is severely impaired. Interestingly, whereas the TRAF6-interacting membrane-proximal domain of CD40 has a minor role in signal transduction, studies utilizing TRAF6 knockout fibroblasts and RNA interference in epithelial cells reveal that the CD40-induced activation of NF-kappaB, JNK, p38, and Akt requires the integrity of TRAF6. Furthermore, we provide evidence that TRAF6 regulates CD40 signal transduction not only through its direct binding to CD40 but also indirectly via its association with TRAF2. These observations provide novel insight into the mechanisms of CD40 signaling and the multiple roles played by TRAF6 in signal transduction.     

Effect of PI3K gene silencing on growth, migration and related proteins expression of CD40 signal-mediated gastric cancer cells

In this study, we investigate effect of PI3K gene silencing on growth, migration and related proteins expression of CD40 signal-mediated gastric cancer cells. We observed that combination of sCD40L with PI3K siRNA could significantly inhibit AGS cells growth, block cells in G1 phase, and promote tumour cells apoptosis after 24 h treatment. Transwell test showed that numbers of cells per visual field in group PI3K siRNA or group sCD40L (after 24 h PI3K siRNA or sCD40L alone treatment) were fewer than that (32.54 ± 4.22) in control group. Numbers of cells per visual field in (after 24 h combination treatment of PI3K siRNA with sCD40L) were significantly fewer than that in group PI3K siRNA or group sCD40L. Compared with group sCD40L, expression level of Fas protein in group sCD40L + PI3K siRNA was significantly increased. The findings suggest that PI3K siRNA may strengthen CD40-induced specific antitumour effect via blocking PI3K/Akt signal pathway, resisting tumour immunoediting regulated by CD40 signal. Combination of sCD40L and PI3K siRNA is an important mechanism of gastric cancer therapy.     

TCR signal transduction in antigen-specific memory CD8 T cells

Memory T cells are more responsive to Ag than naive cells. To determine whether memory T cells also have more efficient TCR signaling, we compared naive, effector, and memory CD8 T cells of the same antigenic specificity. Surprisingly, initial CD3 signaling events are indistinguishable. However, memory T cells have more extensive lipid rafts with higher phosphoprotein content before TCR engagement. Upon activation in vivo, they more efficiently induce phosphorylation of-LAT (linker for activation of T cells), ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), and p38. Thus, memory CD8 T cells do not increase their TCR sensitivity, but are better poised to augment downstream signals. We propose that this regulatory mechanism might increase signal transduction in memory T cells, while limiting TCR cross-reactivity and autoimmunity.     

反效信号传导通路与氧化应激诱导的基因表达

细胞存活与凋亡受生长因子活化的ＥＲＫ与应激活化的ＪＮＫ－ｐ３８信号传导通路二者的动态平衡所控制,ＪＮＫ－ｐ３８通路的持续活化及ＥＲＫ通路的共济失活是细胞凋亡的关键所在。Ｈ２Ｏ２参与多种细胞类型的信号传导,并可诱导早期应答基因的表达。转录因子的激活与抗氧化酶合成有一定的因果关系,但中间环节尚待进一步阐明。     

Hypergravity signal transduction and gene expression in cultured mammalian cells

A number of studies have been conducted during space flight and with clinostats and centrifuges, suggesting that gravity effects the proliferation and differentiation of mammalian cells in vitro. However, little is known about the mechanisms by which mammalian cells respond to changes in gravitational stress. This paper summarizes studies designed to clarify the effects of hypergravity on the cultured human HeLa cells and to investigate the mechanism of hypergravity signal transduction in these cells.     

OX40–OX40 ligand interaction may activate phospholipase C signal transduction pathway in human umbilical vein endothelial cells

Studies have recently supported the emerging role of OX40/OX40L interaction in atherosclerosis. The mechanism of OX40/OX40L interaction may be related to a variety of signal pathways. The most important signal pathway involves the activation of phospholipase C (PLC) which induces diacylglycerol–protein kinase C (DAG–PKC) and the inositol trisphosphate (IP₃)–intracellular free calcium ([Ca²⁺]_i) pathway. The aim of this work was to investigate whether OX40–OX40L interaction can stimulate the PLC signal pathway in human umbilical vein endothelial cells (HUVEC). The DAG and IP₃ level in HUVEC were measured by radio-enzymatic assay. The activity of PKC was detected by its ability to transfer phosphate from [γ-³²P]ATP to lysine-rich histone. [Ca²⁺]_i concentrations were measured by flow cytometric analysis. Results showed that the DAG level was markedly increased in a concentration-dependent, biphasic manner in HUVEC induced by OX40. The early phase was rapid, peaking at 30 s. The late phase reached the maximum level at 15 min and decayed slowly. OX40 increased PKC activity in a dose-dependent manner with two peaks at 40–50 s and 12–16 min, then decreased slowly, yet maintained a high level for at least 30 min. PKC activity was mainly in cytosol at rest and translocated from cytosol to membrane when stimulated by OX40. Similarly, OX40-induced rapid IP₃ formation coincided with the peak of DAG level. Moreover, OX40 also induced peak [Ca²⁺]_i responses including the rapid transient phase and the sustained phase. Anti-OX40L antibody significantly suppressed OX40-induced DAG–PKC and IP₃–[Ca²⁺]_i signal pathway activation in HUVEC. In conclusion, the data suggested that OX40–OX40L interaction induced a robust stimulation of phospholipase C signal transduction pathway in HUVEC.     

The functional expression of calcium-sensing receptors in BRL cells and related signal transduction pathway responsible for intracellular calcium elevation

The calcium-sensing receptors (CaSRs) exist in a variety of tissues and cells. In 2001, Canaff et al. first identified its expression in liver tissue and primary cultured hepatocytes, and demonstrated that GdCl₃ (a specific agonist of CaSR) can cause an increase in intracellular calcium and bile flow. However, authors did not elucidate its mechanisms. Therefore, this study sought to detect CaSR expression in BRL cell line, which is derived from buffalo rat liver, and to reveal the cellular signal transduction pathway by which the CaSR activation results in increased intracellular calcium by BRL cells. In this study, the expression and distribution of CaSR were detected by RT-PCR, Western blotting, and immunofluorescence, and the intracellular calcium concentration [Ca²⁺]_i was measured using LCSM. The results showed that CaSR mRNA and protein were expressed in BRL cells and mainly distributed in cell membrane and cytoplasm. Increased extracellular calcium or GdCl₃ could increase intracellular calcium concentration and CaSR expression. Moreover, this increase of [Ca²⁺]_i could be inhibited or even abolished by U73122 (a specific inhibitor of PLC), 2-APB (an inhibitor of IP₃ receptor), and thapsigargin (an inhibitor of endoplasmic reticulum calcium pump). In conclusion, CaSR is functionally expressed in BRL cells, and activation of CaSR involves in increased intracellular calcium through Gq–PLC–IP₃ pathway.     

Ligation or cross-linking of CD40 has different effects on AGS gastric cancer cells

A gastric cancer (GC) cell line, AGS, has high-level expression of CD40, a tumor necrosis factor receptor (TNFR) family member. CD40 is present on the surfaces of a large variety of cells, including B cells, endothelial cells, dendritic cells and some carcinoma cells, and delivers signals regulating diverse cellular responses, such as proliferation, differentiation, growth suppression, and cell death. In this research, we studied the effects of different forms of CD40 stimulation on AGS cells by flow cytometry, Western blotting and siRNA transfection. We found that different forms of CD40 stimulation, either recombinant soluble CD40L (sCD40L, ligation) or agonist anti-CD40 antibody (cross-linking), induced different effects in AGS gastric cancer cells, proliferation or apoptosis. We also showed that VEGF provided a significant contribution to sCD40L-induced proliferation, while agonist anti-CD40 antibody induced GADD45 upregulation and promoted apoptosis.     

ＩkB激酶及相关信号转导研究进展

ＮＦ－kB（nuclear factor-kB)是一广泛存在的核转录因子,它参与多种基因的转录调控,与许多重要的生理病理过程关系密切。许多细胞外刺可诱导ＮＦ－kB的活性,位点特异的ＮＦ－kB抑制蛋白（ＩkB）的磷酸化对于激活ＮＦ－kB有重要意义。ＩkK(IkB kinase)的发现为进一步了解ＮＦ－kB的功能和调控提供了线索,本文综述了ＩkK的结构、功能及相关信号转导研究进展。     

Maturation of dendritic cells accompanies high-efficiency gene transfer by a CD40-targeted adenoviral vector.

Important therapeutic applications of genetically modified dendritic cells (DC) have been proposed; however, current vector systems have demonstrated only limited gene delivery efficacy to this cell type. By means of bispecific Abs, we have dramatically enhanced gene transfer to monocyte derived DC (MDDC) by retargeting adenoviral (Ad) vectors to a marker expressed on DC, CD40. Adenovirus targeted to CD40 demonstrated dramatic improvements in gene transfer relative to untargeted Ad vectors. Fundamental to the novelty of this system is the capacity of the vector itself to modulate the immunological status of the MDDC. This vector induces DC maturation as demonstrated phenotypically by increased expression of CD83, MHC, and costimulatory molecules, as well as functionally by production of IL-12 and an enhanced allostimulatory capacity in a MLR. In comparing this vector to other Ad-based gene transfer systems, we have illustrated that the features of DC maturation are not a function of the Ad particle, but rather a consequence of targeting to the CD40 marker. This vector approach may thus mediate not only high-efficiency gene delivery but also serve a proactive role in DC activation that could ultimately strengthen the utility of this vector for immunotherapy strategies.     

Comparative analysis of signal transduction by CD40 and the Epstein-Barr virus oncoprotein LMP1 in vivo

There is much evidence, based primarily on in vitro studies, indicating that the Epstein-Barr virus oncoprotein latent membrane protein 1 (LMP1) mimics an activated CD40 receptor. In order to investigate the extent of similarity between LMP1 and CD40 functions in vivo, we analyzed the cytoplasmic signaling properties of LMP1 and CD40 in B cells in a directly comparable manner. For this purpose, we generated transgenic mice expressing either LMP1 or a chimeric LMP1CD40 molecule, which constitutively activates the CD40 pathway, under the control of the CD19 promoter. LMP1 and LMP1CD40 were expressed at similar levels in a B-lymphocyte-specific manner. Similar to LMP1, LMP1CD40 suppressed germinal center (GC) formation and antibody production in response to thymus-dependent antigens, albeit to a greater extent than LMP1. Furthermore, the avidity of the antibodies produced against thymus-dependent antigens was lower for LMP1CD40 transgenic mice than for wild-type and LMP1 transgenic mice. GC suppression was linked to the ability of LMP1CD40 and LMP1 to downregulate mRNA and protein levels of BCL6 and to suppress the activity of the BCL6 promoter. In contrast to LMP1, LMP1CD40 caused an upregulation of CD69, CD80, and CD86 in B cells and a dramatic increase in serum immunoglobulin M. In addition, LMP1CD40 but not LMP1 transgenic mice had elevated numbers of marginal-zone B cells and increased populations of polymorphonuclear cells and/or neutrophils. Consistent with these findings, LMP1CD40 but not LMP1 transgenic mice showed signs of spontaneous inflammatory reactions and the potential for autoimmunity.     

Regulation of the lipopolysaccharide signal transduction pathway by 17beta-estradiol in macrophage cells

We have previously shown that 17beta-estradiol (E2) prevents the activation of brain macrophages, i.e. microglia cells, both in vitro and in vivo. Hormone exerts this inhibitory effect by inhibiting pro-inflammatory gene expression. In this study we further investigated on the molecular mechanism of E2 action in the RAW 264.7 macrophage cell line. We show here that these cells express the alpha-isoform of the estrogen receptor (ERalpha) and not ERbeta. Similarly to its activity in brain macrophages, E2 is able to inhibit the activation program induced by lipopolysaccharide (LPS) in RAW 264.7 cells, as shown by the inhibitory effect of hormone on the morphological conversion and matrix metalloproteinase-9 (MMP-9) expression induced by the endotoxin. In addition, we demonstrate that hormone treatment is not associated with a reduction in the steady-state expression of Toll-like receptor-4 (TLR-4) and CD14, two components of the LPS receptor complex. Our results further confirm the anti-inflammatory role of ERalpha in macrophages and propose that the mechanism of hormone action on macrophage reactivity involves signaling molecules which are down-stream effectors of the LPS membrane receptors.     

Estrogen and progesterone regulate the IL-6 signal transduction pathway in antibody secreting cells

Regulation of the immune response is necessary to allow successful pregnancy. Asymmetric IgG antibodies are involved in fetal maintenance. We have previously demonstrated that estrogen (E2) and progesterone (P4) modulate the synthesis of asymmetric antibodies but the underlying mechanisms remain unclear. Since IL-6 and a progesterone-induced blocking factor (PIBF) were shown to regulate asymmetric antibody synthesis, in this work we analyzed whether E2 and P4 were able to modulate IL-6 signal transduction pathways and the ability of P4 to induce PIBF synthesis, in hybridoma B cells was also evaluated. We found that the IL-6 treatment induced an increase in the expression of gp130 and JAK1 by the hybridoma. E2 and P4 diminished the IL-6-induced gp130 expression in a dose-dependent manner, whereas the expression of JAK1 was not significantly affected. At 10(-6)M concentration, the steroids inhibited the phosphorylation of gp130 and diminished the IL-6-induced STAT3 phosphorylation and traslocation to the nucleus. Maximal PIBF expression was observed when the hybridoma was cultured with 10(-10)M P4, compared to the control (p<0.05). Results demonstrate two molecular mechanisms, the modulation of the IL-6R signal transduction pathway and PIBF induction, which could be involved in the immunoregulatory role of sexual steroids during pregnancy.     

ET-1- and NO-mediated signal transduction pathway in human brain capillary endothelial cells

Previous studies have demonstratedthat functional interaction between endothelin (ET)-1 and nitric oxide(NO) involves changes in Ca²⁺ mobilization and cytoskeletonin human brain microvascular endothelial cells. The focus of thisinvestigation was to examine the possible existence of analogousinterplay between these vasoactive substances and elucidate theirsignal transduction pathways in human brain capillary endothelialcells. The results indicate that ET-1-stimulated Ca²⁺mobilization in these cells is dose-dependently inhibited by NOR-1 (anNO donor). This inhibition was prevented by ODQ (an inhibitor ofguanylyl cyclase) or Rp-8-CPT-cGMPS (an inhibitor of proteinkinase G). Treatment of endothelial cells with 8-bromo-cGMP reducedET-1-induced Ca²⁺ mobilization in a manner similar to thatobserved with NOR-1 treatment. In addition, NOR-1 or cGMP reducedCa²⁺ mobilization induced by mastoparan (an activator of Gprotein), inositol 1,4,5-trisphosphate, or thapsigargin (an inhibitorof Ca²⁺-ATPase). Interestingly, alterations in endothelialcytoskeleton (actin and vimentin) were associated with these effects.The data indicate for the first time that the cGMP-dependent proteinkinase colocalizes with actin. These changes were accompanied byaltered levels of phosphorylated vasodilator-stimulated phosphoprotein, which were elevated in endothelial cells incubated with NOR-1 andsignificantly reduced by ODQ or Rp-8-CPT-cGMPS. The findings indicate a potential mechanism by which the functionalinterrelationship between ET-1 and NO plays a role in regulatingcapillary tone, microcirculation, and blood-brain barrier function.

     

Identification of pathway deregulation - gene expression based analysis of consistent signal transduction

Signaling pathways belong to a complex system of communication that governs cellular processes. They represent signal transduction from an extracellular stimulus via a receptor to intracellular mediators, as well as intracellular interactions. Perturbations in signaling cascade often lead to detrimental changes in cell function and cause many diseases, including cancer. Identification of deregulated pathways may advance the understanding of complex diseases and lead to improvement of therapeutic strategies. We propose Analysis of Consistent Signal Transduction (ACST), a novel method for analysis of signaling pathways. Our method incorporates information regarding pathway topology, as well as data on the position of every gene in each pathway. To preserve gene-gene interactions we use a subject-sampling permutation model to assess the significance of pathway perturbations. We applied our approach to nine independent datasets of global gene expression profiling. The results of ACST, as well as three other methods used to analyze signaling pathways, are presented in the context of biological significance and repeatability among similar, yet independent, datasets. We demonstrate the usefulness of using information of pathway structure as well as genes' functions in the analysis of signaling pathways. We also show that ACST leads to biologically meaningful results and high repeatability.